Betalains and Phenolics in Red Beetroot (Beta Vulgaris)

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Betalains and Phenolics in Red Beetroot (Beta vulgaris) Peel Extracts: Extraction and Characterisation Tytti Kujala*, Jyrki Loponen and Kalevi Pihlaja Department of Chemistry, Vatselankatu 2, FIN-20014 University of Turku, Finland. Fax: +3582-333 67 00. E-mail: [email protected] * Author for correspondence and reprint requests

Z. Naturforsch. 56 c, 343-348 (2001); received January 9/February 12, 2001 Beta vulgaris , Betalains, Phenolics The extraction of red beetroot (Beta vulgaris ) peel betalains and phenolics was compared with two extraction methods and solvents. The content of total phenolics in the extracts was determined according to a modification of the Folin-Ciocalteu method and expressed as gallic acid equivalents (GAE). The profiles of extracts were analysed by high-performance liquid chromatography (HPLC). The compounds of beetroot peel extracted with 80% aqueous methanol were characterised from separated fractions using HPLC- diode array detection (HPLC-DAD) and HPLC- electrospray ionisation-mass spectrometry (HPLC-ESI-MS) tech niques. The extraction methods and the choice of solvent affected noticeably the content of individual compounds in the extract. The betalains found in beetroot peel extract were vulgaxanthin I, vulgaxanthin II, indicaxanthin, betanin, prebetanin, isobetanin and neobetanin. Also cyclodopa glucoside, /V-formylcyclodopa glucoside, glucoside of dihydroxyindolcarboxylic acid, betalamic acid, L-tryptophan, p-coumaric acid, ferulic acid and traces of un identified flavonoids were detected.

Introduction acid in their cell walls (Jackman and Smith, 1996). Phenolic compounds are ubiquitous in the plant Except ferulic acid, also other phenolic acids and kingdom and they have been reported to possess phenolic acid conjugates have been reported in many biological effects. Recently plant phenolics different beetroot materials (Winter and Herr have received interest due to their supposed prop mann, 1986; Bokern et al ., 1991; Waldron et al., erties in promoting human health and the possi 1997; Harborne et al., 1999). The antioxidant activ bility to use them as natural food additives, since ity of beet extracts, especially beet peel extracts, phenolic compounds influence the quality, accept reported recently increase the interest in beetroot ability and stability of foods by acting as flav- compounds (Vinson et al., 1998; Kähkönen et al., orants, colorants and antioxidants (Decker, 1995). 1999). The red beetroot (Beta vulgaris ) has been culti In this paper the effect of extraction method and vated for many hundreds of years in all temperate solvent on the content of red beetroot peel extract climates. Beetroot is used as a vegetable, and its are described. The characterisation of chemical juice and extracts also as traditional medicine, compounds in detail is given for beetroot peel ex food colorant and additive to cosmetics (Henry, tract (80% aqueous methanol) by a high-perfor 1996; Stuppner and Egger, 1996). The use of beet mance liquid chromatography coupled with UV- root as source of colour has focused the beetroot detector, DAD-detector and a mass spectrometer investigations on betalains (red-violet betacyanins equipped with electrospray ion source. and yellow betaxanthins), which are a class of N- containing water-soluble plant pigments. Betalains Materials and Methods are characteristic for the plant order Caryophyl- Chemicals lales (Centrospermae), and there is a mutual ex clusion of betalains and anthocyanins. However, Folin-Ciocalteu’s reagent was purchased from other flavonoids coexist with the betalains (Steg- Fluka (Biochemica, Buchs, Switzerland), sodium lich and Strack, 1990). Many betalain-bearing spe carbonate and L-tryptophan were from Merck cies have relatively large concentrations of ferulic (Darmstadt, Germany) and gallic acid, ferulic acid

0939-5075/2001/0500-0343 $ 06.00 © 2001 Verlag der Zeitschrift für Naturforschung, Tübingen ■ www.znaturforsch.com ■ D 344 T. Kujala et al. • Red Beetroot Peel Extracts and p-coumaric acid were from Sigma (St. Louis, adjusted to 2.0 using HC1. The preparative column MO, USA). All organic solvents used were of (400 x 25 mm I.D., Pharmacia, Uppsala, Sweden) HPLC-grade. filled with Sephadex LH-20 (Pharmacia, Umeä, Sweden) gel was wrapped in aluminum foil to pre Plant material vent exposure to light during the fractionation. The column was washed in advance with water The red beetroots were obtained from a local (pH 2.0) and the extract was transferred on to the farmer (Beta vulgaris , cultivar “Little Ball”). The column. The beetroot peel compounds were beetroots were washed and hand-peeled, and the eluted with acidic water (pH 2.0), neutral water collected peels were cut into small pieces and and different strengths (20-100%) of aqueous stored at -25° C until lyophilisation. Lyophilised methanol. The first fraction contained sugars and plant material was ground with a mortar and pes was discarded. The elution was continued and tle. fractions (about 100 ml per fraction) were col lected. The fractions were taken almost to dryness Extraction (analytical) by a rotary evaporator and the residues were di Three replicates were prepared with both ex luted to water in prior to HPLC and HPLC-ESI- traction methods and solvents. MS analyses. Portion of the fractions were lyo M ethod A: 500 mg of lyophilised material was philised for further analyses. extracted with 25 ml of solvent (80% aqueous methanol or water) in planar shaker (Promax Determination of total phenolics 2020, Heidolp, Germany) for 45 min. The homoge- The content of total phenolics in the extracts nate was centrifuged for 10 min (1500 xg) and the was determined according to a modification of the clear supernatant was collected. The insoluble part Folin-Ciocalteu method (Nurmi et al., 1996) on a was re-extracted twice with 25 ml of solvent for 45 Perkin-Elmer Lambda 12 UV/VIS Spectrometer and 20 min. The combined extracts were taken to (Norwalk, CT, USA). The total phenolic content dryness by a rotary evaporator and the residue was expressed as gallic acid equivalents (GAE) in was dissolved in 20 ml of water. milligrams per gram of dry plant material. M ethod B\ 500 mg of lyophilised material was homogenised (Ultra-Turrax T 25, Janke & Kunkel, HPLC analysis IKA-Labortechnik, Germany) for one min with 10 ml of solvent (80% aqueous methanol or Analytical chromatographic measurements water). The homogenate was centrifuged for were performed on an HPLC system consisting of 10 min (1500xg) and the clear supernatant was a Merck-Hitachi L-7200 autosampler, Merck-Hi- collected. The insoluble part was re-extracted with tachi L-7100 HPLC pump, Merck-Hitachi L-7400 10 ml of solvent. The combined extracts were UV/VIS detector and Merck-Hitachi D-7000 taken to dryness by a rotary evaporator and the interface (all from Hitachi, Tokyo, Japan). The ab residue was dissolved in 20 ml of water. sorption spectra (240-370 nm) of individual com pounds of extracts and fractions were recorded on a system consisting the pump and LC-235 diode Extraction (preparative) and fractionation array detector (Perkin-Elmer, Norwalk, CT, USA) Prior to the HPLC-ESI-MS analysis a concen connected to a Graphic Printer GP-100 (Perkin- trated beetroot peel extract was prepared. The ex Elmer, Beaconsfield, UK). The compounds were tract was prepared by a modification of the analyt detected by an HPLC method developed earlier ical method B, the initial amount of dry beetroot in our laboratory (Ossipov et al., 1995). The col peel being 20 g and the volume of used solvent, umn used was a LiChrocart RP-18 (250 x 4.0 mm 80% aqueous methanol, 800 ml. The combined ex I.D., Purospher, 5-|.im, Merck, Darmstadt, Ger tracts were taken to dryness and the residue was many) equipped with a precolumn. The injection dissolved in 40 ml of water. volume was 20 |.il. A constant flow rate was 1.0 ml The concentrated extract was centrifuged for min-1. The eluents were acetonitrile (A) and for 10 min (1500xg) and the pH of the extract was mic acid - water (5:95, v/v) (B). The elution pro T. Kujala et al. • Red Beetroot Peel Extracts 345 file was: 0-5 min, 100% B (isocratic); 5-60 min, The mass spectrometer was operated in the 0-30% A in B (linear); 60-70 min, 30-60% A in negative and positive ion modes. Mass data were B (linear). In order to define the composition of acquired by the scan mode, consisted of scanning beetroot peel extract, both betalains and pheno from m /z 100 to 1100 with 0.30 amu steps. The lics, 280 nm was chosen as detection wavelength, spray needle potential for the negative ion experi after also wavelenghts 320, 365 and 520 nm were ments was -4000 V. Orifice plate voltage was set tried. at - 35 V. Ring voltage was set at -220 V. Nebu lizer gas (purified air) value was set to value 8 and curtain gas (N2) to value 10. Settings for the posi HPLC-ESI-MS analysis tive ion experiments were: the spray needle poten A high-performance liquid chromatographic- tial, 5200 V; orifice plate voltage, 45 V; ring volt mass spectrometric analysis of red beetroot peel age, 220 V; nebulizer gas, value 8 and curtain gas, compounds were carried out with a Perkin-Elmer value 10. Heated nitrogen gas temperature was SCIEX API 365 triple-quadrupole mass spectrom 300 °C. eter (Sciex, Toronto, Canada) incorporated to a Perkin-Elmer Series 200 HPLC system and Apple Results and Discussion Macintosh 8.1 data system. The ion source was an ionspray (pneumatically assisted electrospray ioni sation option; Sciex, Toronto, Canada). The HPLC The total phenolic contents of the red beetroot system was comprised of two Perkin-Elmer Series peel extracts prepared by different extraction 200 micro pumps (Perkin-Elmer, Norwalk, CT, methods and solvents decreased in the order 80% USA) and a 785A UV/VIS detector (Perkin-El aqueous methanol extract prepared by method A mer, Norwalk, CT, USA). Samples were intro (24.1 ± 0.3 mg/g GAE), water extract prepared by duced into the system by a Perkin-Elmer Series method A (20.5 ± 0.4 mg/g GAE), 80% aqueous 200 Autosampler (Perkin-Elmer, Norwalk, CT, methanol extract prepared by method B (18.8 ± USA). The separation of individual compounds 0.3 mg/g GAE) and water extract prepared by was achieved on a column mentioned earlier. method B (17.4 ± 0.4 mg/g GAE). The extraction The HPLC solvent system consisted of acetoni- method and solvent were found to affect notice trile (A) and formic acid - water (0.4:99.6, v/v) ably the content of individual compounds in beet (B). The elution profile was: 0-5 min, 100% B root peel extract on the basis of HPLC analyses. (isocratic); 5-50 min, 0-20% A in B (linear); 50- The area of neobetanin peak in the 80% aqueous 60 min, 20-70% A in B (linear). The injection vol methanol extracts made by method A and the area ume was 20 [il. The flow rate was 1.0 ml min 1 of cyclodopa glucoside peak in the 80% aqueous before the ESI. The main part of the flow was split methanol extracts made by method B, were and only minor part (200 [il min-1) was introduced multiple compared with the other extracts. The into the ion source. area of isobetanin peak in the water extracts made

□ betanin □isobetanin □neobetanin

4000000 3500000 3000000 2500000 2000000 1500000 1000000 500000 * |* | 0 Fig. 1. Areas of betanin, isobetanin and neobe tanin peaks in the chromatograms of red beet Method A, Method A, Method B, Method B, root peel extracts prepared with two different 80% MeOH water 80% MeOH water extraction methods and solvents. 346 T. Kujala et al. • Red Beetroot Peel Extracts by method A was greater than in other extracts in Table I. Mass spectral data (negative and positive detec regard to the area of betanin peak (Fig. 1). tion) of beetroot peel constituents. Four betacyanins (betanin, prebetanin, isobe- Structure attribution m/z Ions tanin, neobetanin), three betaxanthins (vulgaxanthin I, vulgaxanthin II and indicaxanthin), cyclo- Cyclodopa glucoside 356 [M-H]- 713 [2M-H] dopa glucoside, TV-formylcyclodopa glucoside, 358 [M+H]+ betalamic acid, L-tryptophan, p-coumaric acid and 715 [2M+H]+ ferulic acid were found in the water fractions of N-Formylcyclodopa 383 [M-H]- crude extract of beetroot peel. Glucoside of dihy- glucoside 767 [2M -H]- droxyindolcarboxylic acid was found in the first 385 [M+H]+ methanolic fraction of crude extract. The identifi Vulgaxanthin I 338 [M-H]- cation of these compounds was based on their mo 677 [2M -H]- lecular weights defined from HPLC-ESI-MS 340 [M+H]+ analyses and the comparisons of the UV-spectral characteristics of the corresponding peaks with Vulgaxanthin II 339 [M-H]- 341 [M+H]+ standards or literature data. The UV-spectral char acteristics of L-tryptophan, p-coumaric acid and Indicaxanthin 307 [M -H]- ferulic acid were compared with standards, and Betalamic acid 210 [M-H]- those of other compounds with literature data (be 421 [2M-H]- tanin, isobetanin, vulgaxanthin I, vulgaxanthin II 212 [M+H]+ and betalamic acid (Musso, 1979); indicaxanthin Betanin 549 [M-H]- (Piattelli et al ., 1964); prebetanin (Wyler et al., 551 [M+H]+ 1967); neobetanin (Alard et al., 1985); cyclodopa glucoside, TV-formylcyclodopa glucoside and gluco Glucoside of dihydroxyindol 192 [M-H-glucose]_ carboxylic acid 354 [M-H]- side of dihydroxyindol carboxylic acid (Wyler et 709 [2M -H]- al., 1984)). Vulgaxanthin I and vulgaxanthin II are known to cooccur in beetroot (Steglich and Strack, L-Tryptophan 203 [M-H]- 407 [2M -H]- 1990). The vulgaxanthins coeluted in the HPLC 205 [M+H]+ analyses, but on the basis of the HPLC-ESI-MS 409 [2M+H]+ analyses vulgaxanthin I was found to be predomi Prebetanin 629 [M-H]- nant. On the basis of UV-spectral characteristics 389 [betanidin +H]+ also flavonoids were found in the methanolic frac 631 [M+H]+ tions. The mass spectral data of red beetroot peel con Isobetanin 549 [M-H]- 551 [M+H]+ stituents are listed according to the retention order (analytical column) in Table I. Negative- and posi Neobetanin 547 [M-H]- tive ion mass spectra of components of the extract 549 [M+H]+ allowed the definition of molecular masses. Except p-Coumaric acid 163 [M-H]- indicaxanthin and glucoside of dihydroxyindolcar- 327 [2M-H]" boxylic acid, each compound gave clear [M-H]- 165 [M+H]+ ions and [M+H]+ ions in the negative and positive Ferulic acid 193 [M-H]- modes, respectively, with relatively high intensity. 195 [M+H]+ Glucoside of dihydroxyindolcarboxylic acid and prebetanin were the only compounds in this study, which gave detectable fragment ions. Glucoside of most spectra of the components, additional infor dihydroxyindolcarboxylic acid (M.W. 355) showed mation in the forms of [2M-H]~ or [2M+H]+ ions fragment ion at m /z 192 with negative ion mode were detected. indicating the loss of glucose residue. Prebetanin Betanin is found as the predominant colouring (M.W. 630) showed fragment ion [betanidin + H]+ in most varieties of beetroot (Henry, 1996). Other at m /z 389 with positive ion mode. Moreover, in betacyanins reported in red beetroot materials are T. Kujala et al. ■ Red Beetroot Peel Extracts 347 betanidin, isobetanidin, isobetanin, prebetanin, acid, ferulic acid dehydrodimers, p-hydroxyben- neobetanin, amaranthin, lampranthin I and lam- zoic acid, vanillic acid and trans-p- coumaric acid pranthin II (Alard et al., 1985; Pourrat et al., 1988; in cell walls of beetroot. Bokern et al. (1991) found Bokern etal., 1991; Jackman and Smith, 1996). The five ferulic acid conjugates from cell cultures of solvents used to extract betalains are mainly water beetroot, when freeze-dried cells were extracted and aqueous methanol (50-80%). Degradation with 50% aqueous methanol. Ferulic acid has also products of betanin are isobetanin, decarboxylated been found in the leaves of Beta vulgaris (H ar betanin, betalamic acid and cyclodopa glucoside borne et a l, 1999). Winter and Herrmann (1986) (Jackman and Smith, 1996). Except decarboxyl reported several phenolic acids from different ated betanin, all these compounds were detected parts of beetroot extracted with 80% aqueous in the fractions of crude extract. The natural oc methanol (3'-caffeoylquinic acid, 3'-p-coumaro- currence of betalamic acid and cyclodopa gluco ylquinic acid, 3'-feruloylquinic acid, 1-O-p-coum- side has been reported (Wyler et a l, 1984; Steglich aroyl-ß-D-glucose, l-O-feruloyl-ß-D-glucose, l-O- and Strack, 1990). The occurrence of free cyclo sinapoyl-/?-D-glucose). Also dihydrocaffeic acid dopa glucoside in red beetroot has also been re has been detected in beetroot (Harborne et a l, ported to support its role as intermediate of be 1999). tanin biosynthesis. The detected N- L-Tryptophan is found as a protein amino acid formylcyclodopa glucoside and glucoside of dihy- in all plants, but it is often present in such low droxyindolcarboxylic acid have been earlier re amounts that it can not be detected readily (Har ported in air exposed solutions of cyclodopa gluco borne et a l, 1999). The results of Kato-Noguchi side (Wyler et a l, 1984). 14,15-dehydroxybetanin, et al. (1994) suggest that L-tryptophan may be an neobetanin, has been attributed to be an artefact allelochemical, which affects the growth or germi formed during the isolation process (Wyler, 1986), nation of different plant species. but also its natural occurrence has been proved (Strack et a l, 1987). Acknowledgements Although red beetroot has been reported to contain many phenolic acids, only p-coumaric acid This work was supported by the Finnish Minis and ferulic acid were detected in the fractions of try of Agriculture and Forestry and by Tekes Tech peel extract. Waldron et al. (1997) found ferulic nology Development Center. 348 T. Kujala et al. ■ Red Beetroot Peel Extracts

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